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Protein phophorylation is a widespread post-translational modification that plays a crucial role in many cellular processes. However the analysis of phosphopeptides and reproducibility of methods is not easy because of many factors, among which relatively low abundance makes it a challenging analytical process.
We present an automated method for the phophopeptide analysis using an immobilized metal affinity chromatography (IMAC) column to enrich phosphopeptides and subsequent reverse-phase HPLC analysis. The recently developed IMAC is an analytical HPLC column that can be used repeatedly and reproducibly. By using a dual pump and automatic sample injections, the entire phophopeptide analysis is automated and is completed in about an hour.
We used tryptic digestions of beta-casein as the model to evaluate nonspecific binding of some nonphosphorylated and all phosphorylated peptides to the IMAC column under the loading conditions (20 mmol/L formic acid). Under the release conditions at pH 9 with ammonium formate, all the expected phosphopeptides are released and recovered quantitatively. The data are presented to show the specificity, recovery, and reproducibility of the method.